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Results

JVR was strongly associated with increased normalized WB (p = 0.014) and GM (p = 0.002) volumes across all three subject groups. There was a trend towards increased
WB and GM volumes, which was accompanied by decreased CSF volume, in the JVR-positive
subjects in both the MCI and AD groups. When the MCI and AD subjects were aggregated
together significant increases were observed in both normalized WB (p = 0.009) and GM (p = 0.003) volumes for the JVR-positive group. No corresponding increases were observed
for the JVR-positive subjects in the control group. Through receiver operating characteristic
analysis of the brain volumetric data it was possible to discriminate between the
JVR-positive and negative AD subjects with reasonable accuracy (sensitivity = 71.4%;
specificity = 88.9%; p = 0.007).

Conclusions

JVR is associated with intracranial structural changes in MCI and AD patients, which
result in increased WB and GM volumes. The neuropathology of this unexpected and counterintuitive
finding requires further investigation, but may suggest that JVR retrogradely transmits
venous hypertension into the brain and leads to brain tissues swelling due to vasogenic
edema.

Keywords:

Background

Alzheimer's disease (AD), the most common form of dementia in the elderly, is thought
to be caused by an imbalance between amyloid-β (Aβ) production and clearance leading
to Aβ accumulation in the central nervous system (CNS), which causes neuronal damage
and death, manifesting as progressive clinical dementia [1-3]. It has been shown that patients with AD have 30% slower clearance of Aβ [1]. One of the possible etiologies of decreased Aβ clearance is impaired cerebrospinal
fluid (CSF) flow [1,4]. When venous hypertension occurs in the superior sagittal sinus, CSF absorption is
also impaired, leading to altered CSF outflow [5-7]. Jugular venous reflux (JVR) characterized by a retrograde flow in internal jugular
veins (IJVs) during Valsalva-like manoeuvres (VM) or spontaneously, is found more
frequently in the elderly [8,9]. Studies have shown that JVR can retrogradely transmit hypertension into the cerebral
venous system and that it is associated with white matter (WM) abnormalities in the
elderly [10,11]. Consequently, there is reason to believe that JVR might alter CSF absorption, and
that this in turn might influence the clearance of Aβ. Given this, we hypothesized
that JVR might be associated with mild cognitive impairment (MCI) and AD, and that
this association might manifest itself in structural changes in the brain parenchyma.
To this end, we undertook a case-controlled study to explore the issue by evaluating
the relationship between JVR and global and tissue specific brain parenchyma volumetric
parameters. Volumetric analysis of brain parenchyma structures measured on serial
magnetic resonance imaging (MRI) scans has been shown to provide an objective and
quantitative method for examining neuropathological changes associated with AD [12-18].

Methods

Patient population

Between December 2008 and April 2010, Taiwanese residents consecutively admitted to
a memory clinic at Taipei Veterans General Hospital, Taiwan due to subjective memory
complaints were assessed for inclusion in this study. Neurologists performed clinical
and neurologic evaluations of all participants. Standard neuropsychological assessments,
including the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating (CDR)
scale were used.

Subjects eligible for participation in the current study were 55 years of age or older,
had a CDR score ≤1 (as an assurance that they could cooperate during the Valsalva
manoeuvre for JVR detection), and were willing to receive brain MRI and neck duplex
ultrasonography. Exclusion criteria for all subjects were a past history of stroke,
ischemic heart disease, congestive heart disease, valvular heart disease, cardiac
arrhythmia, pulmonary diseases, or malignancy, and having brain MRI of insufficient
quality for performing quantitative brain volumetric analysis. The inclusion/exclusion
criteria, clinical evaluation, and duplex ultrasonography and MRI protocols and rating
method, were pre-defined before the study.

Vascular risk factors were defined according to international guidelines and prospectively
identified using all available information including medical charts, laboratory results,
patient interviews, and neurological examinations. Hypertension was defined as a history
of hypertension, use of antihypertensive medications, or a measured blood pressure
consistently >140/90 mmHg. Hyperlipidemia was defined as a cholesterol level >200 mg/dL,
low density lipoprotein >150 mg/dL, triglyceride level >150 mg/dL, or history of hyperlipidemia.
Diabetes was defined as a history of diabetes, use of medications for diabetes, or
an elevated fasting blood glucose >126 mg/dL.

Subjects were classified in AD, mild cognitive impairment (MCI) or control groups
according to the criteria of National Institute of Neurological and Communicative
Disorders and Stroke/Alzheimer's Disease and Related Disorders Association [19], and by Petersen et al.’s study revised by the Stockholm consensus group [20,21]. The hospital’s Institutional Review Board approved the study and each included participant
or his/her caregiver provided informed consent.

Color-coded duplex ultrasonography for JVR determination

Neck color-coded duplex sonography was performed in all subjects with a 7-MHz linear
transducer (iU22; Philips, New York, NY, USA) by the same technician, who was blinded
to subjects’ characteristics. On examination, subjects were in a head-straight, flat
supine position after a quiet 10 min rest. The IJV was initially insonated longitudinally
and thoroughly from the proximal part of the neck base rostrally to the distal part
at the submandibular level in order to detect any possible spontaneous JVR at baseline.
Then, the VM was performed by forcible expiration from subject’s mouth into a flexible
rubber tube connected to a manometer. Subjects were asked to reach 40 mmHg Valsalva
pressure and maintain it for at least 10 seconds. During the VM, the distal margin
of the window of the color signal was placed at the tip of the flow divider of the
internal carotid artery. The color box was adjusted to include the entire lumen of
the IJV; if retrograde color appeared in the center of the lumen, the retrograde flow
would then be confirmed by Doppler spectrum. JVR was determined when the retrograde-flow
color in the center of the lumen and the Doppler-flow waveform demonstrated reversal
of flow for more than 0.5 seconds [9-11]. JVR was deemed to have occurred if it could be detected spontaneously at baseline
or during the VM. The subjects were classified according to JVR status: subjects with
no JVR on both sides were classified as JVR-negative, and subjects with JVR detected
on either or both sides during VM, or spontaneously, were graded as being JVR-positive.

MRI analysis

The MRI volumetric analyses were blinded to the subjects’ demographic and clinical
characteristics. For brain extraction and tissue segmentation into normalized whole
brain (WB), gray matter (GM), WM, and CSF volumes, the SIENAX cross-sectional software
tool was used (version 2.6), with corrections for T1-hypointensity misclassification
using an in-house developed in-painting program, as previously described [22].

Statistical analysis

Statistical analysis was undertaken using a combination of the Statistical Package
for Social Sciences (SPSS, IBM, Armonk, New York, USA) and in-house algorithms written
in Matlab (Mathworks, Natick, Mass) with the aim of evaluating the impact of JVR on
the respective MRI variables.

Parametric (one-way ANOVA) and non-parametric (2-tailed Mann Whitney U-test, chi square
test) univariate analyses were performed on the respective study cohorts to identify
significant differences between the various groups. Values of p < 0.05 were considered
statistically significant. For the purposes of this analysis, individuals were simply
classified according to clinical disease classification (i.e. controls, MCI and AD)
and whether or not they were JVR-positive.

In order to calculate sensitivity and specificity scores related with any structural
MRI changes that might be associated with JVR, we also performed receiver operating
characteristic (ROC) analysis using a bespoke Matlab algorithm [23]. So as to maximize the discrimination characteristics of the ROC analysis, principal
component analysis (PCA) was used to combine MRI variables identified as being influential
by the univariate analysis. The ROC analysis was then performed using the first principal
component (i.e. the principal component responsible for most variance in the data).

Demographic and clinical univariate analysis

Table 1 shows the comparisons of clinical characteristics and MRI variables between control,
MCI and AD groups. From this it can be seen that for all but three of the clinical
variables there was no significant difference between the respective groups. The only
exceptions to this were: the MMSE score, which was significantly lower in the AD group
(p < 0.001); the number of years in education, which was on average approximately 3 years
less in the MCI and AD groups (p = 0.018); and hyperlipidemia, which had a higher incidence in the AD group (p = 0.015). Of the MRI variables, only normalized WB volume showed a significant difference
between the three groups, being significantly smaller in the AD group (p = 0.034).

Table 2 shows demographic, clinical and MRI characteristics of the whole study population
aggregated together and grouped according to JVR status (i.e. JVR-positive and negative).
The two JVR-graded groups were closely age-matched and had similar clinical characteristics,
with no significant differences in sex, education, and disease classification. However,
significantly increased normalized WB (p = 0.014) and GM (p = 0.002) volumes were observed in the JVR-positive group. The increase in brain parenchyma
volume in the JVR-positive subjects was matched by a corresponding decrease in CSF
volume, although this did not reach significance.

Table 2.Demographic and clinical characteristics of study cohort grouped by JVR status (i.e.
positive or negative) for all groups aggregated together

In order to determine whether or not the increase in WB and GM volumes was exhibited
in all three clinical groups, we repeated the univariate analysis for each disease
classification group. The results of this analysis are presented in Table 3, which reveals a trend towards increased WB and GM volumes in the JVR-positive subjects
in both the MCI and AD groups, evidenced by Cohen’s d effect sizes >0.8. When the
MCI and AD subjects were aggregated together the univariate analysis revealed even
more significant increases in both normalized WB (p = 0.009) and GM (p = 0.003) volumes for the JVR-positive group. No corresponding difference was observed
between the JVR-positive and negative subjects in the control group. Similarly in
the controls, no significant difference in CSF volume was observed between the JVR-positive
and negative groups, whereas in the MCI and AD subjects there was a trend towards
reduced CSF volume.

Table 3.MRI variables classified according to JVR status (i.e. positive or negative) for each
disease group

Separate analysis of the JVR-negative group revealed a statistically significant difference
between the controls and the MCI and AD subjects for the normalized WB (p = 0.023) and WM (p = 0.028) volumes, both of which were greatly reduced in the JVR-negative MCI and
AD subjects. By comparison, no corresponding reductions in brain parenchyma volume
were observed in the JVR-positive MCI and AD subjects compared with the JVR-positive
controls.

Receiver operating curve analysis

The results of the univariate analysis revealed JVR to be associated with a trend
towards increased WB and GM volumes in both the MCI and AD groups, something that
was not observed in the control group. In order to confirm this finding we used PCA
to orthogonalize/combine these variables and used the resulting first principal component
to perform a ROC analysis, the results of which are presented in Figure 1 and Table 4. From this, it can be seen that the ROC results are strongly significant for the
MCI and AD groups, and appear to corroborate the findings of the univariate analysis.
While the ROC analysis did not yield a significant result for the control group, it
was able to discriminate between the JVR-positive and negative subjects in the other
two groups with reasonable accuracy (>70%). Indeed, for the AD group, the ROC analysis
achieved sensitivity and specificity scores of 71.4% and 88.9%, respectively (p = 0.007). As such, the results suggest that JVR was associated with structural changes
in the brain parenchyma in both the MCI and AD subjects.

Discussion

The results of the study do not support the hypothesis that JVR is specifically associated
with MCI and AD. The incidence of JVR was very similar in both the control and MCI
groups, and was actually lower in the AD group. Having said this, the results suggest
that JVR is associated with a rather unexpected phenomenon. JVR appears to be associated
with structural changes in the brain parenchyma of patients with MCI and AD that were
not observed in the control group. This is highlighted in the results presented in
Table 3, which revealed a marked difference in response to JVR between the controls and the
other two groups. Overall, the subjects with JVR had greater WB and GM volumes, which
was accompanied by decreased CSF volume, compared with those without JVR (Table 2). This effect was particularly marked in the MCI and AD groups, whereas it was absent
in the controls (Table 3). As such, the observation that AD patients with JVR exhibit larger WB volumes is
a surprising finding, as AD is normally characterized by advanced brain atrophy. This
can be clearly seen if one analyzes the JVR-negative and positive groups separately.
In the JVR-negative subjects there was a statistically significant reduction in global
and tissue specific brain parenchymal volumes in the AD and MCI groups compared with
the controls - a finding that is consistent with the observations of many other researchers
[12-15,24]. However, no corresponding reduction was observed in the JVR-positive group, implying
that in some way JVR inhibited brain volumetric loss in the AD and MCI subjects. Compared
with insufficient cerebral arterial supply (arterial ischemia), cerebral venous drainage
impairment with venous hypertension causes more severe vasogenic edema and brain–blood
barrier damage [5-7]. Previous studies of JVR provide evidence that retrograde-transmitted venous hypertension
from JVR can reach the cerebral venous system [10,11]. It is therefore possible that JVR retrogradely transmits venous hypertension into
the brain, leading to increased permeability of the blood–brain barrier (BBB), resulting
in vasogenic edema, causing the brain tissue to swell. Disruption of the BBB will
allow plasma molecules to pass into the brain, with the result that an osmotic pressure
gradient is established which will contribute to edema formation. While it is not
known if this mechanism is at work, it is noticeable that JVR was associated with
a marked reduction in CSF volume in the MCI and AD subjects, something that would
be consistent with an influx of CSF into the parenchymal tissue. Alternatively, JVR
might promote the retention of blood in the cerebral veins [25] – something that might increase the volume of the brain parenchyma.

The results of the ROC analysis demonstrate that it is possible to discriminate between
the JVR-positive and negative MCI and AD subjects with reasonable accuracy using just
the MRI variables, normalized WB volume and normalized GM volume, whereas this was
not the case in the control group. As such, this finding appears to corroborate those
of the univariate analysis. Furthermore, the ROC analysis suggests that a progressive
effect may be occurring, which is stronger in the AD group (area under curve (AUC) = 0.794;
p = 0.007) than in the MCI group (AUC = 0.713; p = 0.012). While this finding is difficult to interpret, it is known that MCI is frequently
a precursor to AD [26].

While the exact physiological mechanisms behind our intriguing observations are unclear,
it is known that JVR can induce hypertension in the dural sinuses [10,11] and that this can alter intracranial CSF dynamics [27]. Therefore, it may be that retrograde-transmitted venous pressure associated with
JVR inhibits CSF absorption into the superior sagittal sinus [28]. Absorption of CSF into the dural venous sinuses requires a pressure gradient of
about 5–7 mmHg [29,30]. Therefore, an increase in venous pressure of few mmHg due to occlusion of the venous
drainage pathways [31], or reflux, will tend to inhibit the bulk flow of CSF, as observed by Zamboni et
al. [32]. If CSF flow is inhibited, then this might result in increased biochemical concentrations
in the CSF. Overproduction of Aβ is thought to damage WM in AD [33]. Given that patients with AD have been shown to exhibit 30% slower Aβ clearance [1], it has been postulated [4] that accumulation of Aβ in the CSF, arising from venous hypertension, may precipitate
the onset of AD. However, our results do not support this conclusion. While JVR may
be associated with accumulation of Aβ in the CSF, our results do not indicate that
JVR precipitates the onset of AD. Indeed, the majority of AD patients in our study
were JVR-negative. Having said this, the results suggest that JVR is having an effect
on the brain parenchyma of the MCI and AD patients, and the possibility that this
might involve CSF accumulation of Aβ cannot be excluded. Further investigations are
therefore needed to elucidate the underlying neuropathological mechanisms associated
with our observations.

In this study we focused solely on JVR and ignored other phenomena associated with
constricted cerebral venous outflow [34]. However, it may be that restricted venous outflow, such as that associated with
chronic cerebrospinal venous insufficiency [35,36], might also be influential. Studies of cerebral arteriovenous malformation have shown
that the elevated venous pressure and its insults to intracranial structures are more
severe when combined with obstruction in other venous outflow tracts [37,38]. Therefore it may be the case in JVR, that retrogradely-transmitted venous pressure
into cerebral circulation needs additionally an obstruction of contralateral venous
outflow pathway to cause significant venous hypertension and consequently intracranial
insults. It is important to remember that a diagnosis of ‘no-JVR’ does not preclude
the possibility that constricted cerebral venous outflow might be present. Furthermore,
engorged veins are frequently observed upstream of stenotic lesions [39] and it may be that muscular compression of these veins also contributes to JVR.

Although it yielded novel and interesting findings, it should be noted that our study
was limited in its scope, having a relatively small sample size. In particular, the
AD group contained fewer JVR-positive individuals compared with the other two groups.
Also, because of the limited numbers involved, we restricted ourselves to a simple
positive/negative JVR classification and did not distinguish between bilateral, left
and right-sided JVR. It is therefore not known the extent to which left and right
sidedness in JVR influences brain atrophy and further work will be required to evaluate
this. Furthermore, there are other venous abnormalities associated with aging and
other neurological disorders which we were not able to assess [9,27,40]. Nevertheless, our findings are novel and suggest that cerebral venous drainage impairment
may influence the neuropsychology of AD. Further studies, particularly longitudinal
studies, are therefore needed to build on our initial findings.

Conclusions

JVR is associated with intracranial structural changes in MCI and AD patients, which
result in increased WB and GM volumes. Although the neuropathology associated with
this unexpected and counterintuitive finding requires further investigation, it may
be that JVR retrogradely transmits venous hypertension into the brain, and that this
leads to the brain tissues swelling due to vasogenic edema.

Competing interests

The authors declare that they have no competing interests regarding study in question.
Robert Zivadinov received personal compensation from Teva Pharmaceuticals, Biogen
Idec, EMD Serono Claret and Genzyme for speaking and consultant fees. Dr. Zivadinov
received financial support for research activities from Biogen Idec, Teva Pharmaceuticals,
Genzyme and Novartis. Clive Beggs, Niels Bergsland, Simon Shepherd, Michael Dwyer,
Chih-Ping Chung, Pei-Ning Wang and Han-Hwa Hu have nothing to disclose.

Authors’ contributions

CBB, CPC, NB, PNW, SJS, CYC, MGD, HHH, and RZ have made substantial contributions
to conception and design, or acquisition of data, or analysis and interpretation of
data. CBB, CPC and RZ have been involved in drafting the manuscript, while NB, PNW,
SJS, CYC, MGD and HHH revised it critically for important intellectual content. All
authors have given final approval of the version to be published.

Acknowledgements

This work has been supported in part by grants from the Annette Funicello Research
Fund for Neurological Diseases and Jacquemin Family Foundation.